In the post-genomics era, the rate of data generation is far outpacing scientists' ability to analyze them. In the area of protein function and interactions, there is an ever-expanding catalog of posttranslational modifications and an analogous library of enzymes catalyzing the corresponding transformations to be studied. Many of these enzyme-substrate interactions are believed to play key roles in mediation diseases processes. Therefore, a proper understanding of the interactions between the enzymes and their cognate substrates promises to provide fundamental breakthroughs for new treatments and therapies. However, to study these interactions, it is of critical importance to have efficient tools that can readily identify from among many possible substrate candidates the biologically relevant substrates and measure the enzymatic activities of the enzymes. In the past, the lack of such tools has greatly hindered progress in our understanding of these fundamental biological processes.
In recent decades, fluorescent probes have emerged as a very useful class of research tool for biological and biochemical research. They are particularly useful in enzyme assays and the study of molecular trafficking and interactions. For example, fluorescein is commonly attached to biologically active molecules (e.g. antibodies) as a tag to track the movements of these molecules. Other fluorogenic and calorimetric substrates have also been used to assay protease activity and substrate specificity (Neefies et al., Nature Rev. Drug Discovery 3, 58-69 (2004), the entire content of which is incorporated herein in by reference) and have been used to a lesser extent to assay the activity of many other enzymes including glycosidases, phosphatases, sulfatases and esterases (Haugland, R. P. Handbook of Fluorescent Probes and Research Products, Molecular Probes Inc., Eugene, Oreg., 2002; Baruch et al., Trends in Biotech, 14, 29-35 (2004), the relevant portions of which are incorporated herein by reference).
Despite their increasing popularity, there remain many limitations to flouorescent probes. As a general rule, no fluorescent probes are universally applicable and different types of probes must be developed to meet different application requirements. For instance, the above-mentioned fluorescent tags, while useful as molecular beacons, have several major limitations for studying enzymatic activities. One major disadvantage is that attaching a bulky molecule such as fluorescein to a protein may disrupt the protein's native conformation, thereby, disrupting the very interaction which the experiment attempts to observe. Furthermore, many substrates of enzymes are peptides. Often, the functional groups of a peptide suitable for making covalent attachment to the fluorescent tags (e.g. the N and C termini, the carboxyl group of Asp side-chain, etc.) are also important in interacting with the enzyme. This poses a difficult challenge for adding fluorescent tags to peptide substrates, as attachment of the fluorescent tags would likely interfere with the native interactions of the peptides. Thus, there still exists a need for more adaptable fluorogenic probes that can easily be incorporated into peptides with minimal structural and activity impact for probing enzymatic activities (Goddard et al., Trends in Biotech, 22, 363-370 (2004), the entire content of which is incorporated herein by reference).
One excellent example of a family of enzymes whose study would greatly benefit from such a probe is the protein tyrosine phosphatase (PTP) enzyme family. The PTPs are a diverse family of enzymes involved in key signaling pathways and have been implicated in a number of disease states (Zhang et al., PNAS, 90, 4446-4450 (1993); Alonso et al., Cell, 117, 699-711 (2004), the relevant portions of which are incorporated herein by reference). Recent work indicates that PTPs have distinct substrate specificities, but a detailed understanding has been slow to emerge because the chemical tools necessary for studying PTP activity against peptide substrates were not available. Up until recently, assays for phosphatase activities have employed fluorogenic substrates based on para-nitrophenylphosphate (pNPP) and 4-methylumbelliferone (4-MU). These substrates suffer from the problem that maximum fluorescence of the reaction product requires an alkaline pH, thus, their sensitivity are highly influenced by the pH and prone to error (Montalibet et al., Methods, 35, 2-8 (2005), the entire content of which is incorporated herein by reference).
Alternative approaches such as mass-spectroscopy (Wang et al., Biochem., 41, 6202-6210 (2002), the entire content of which is incorporated herein by reference) and protease-coupled assays (Nishikata et al., Biochem. J. 343, 385-391 (1999), the entire content of which is incorporated herein by reference) have been reported in the art. These approaches, while more accurate than measuring the fluorescence changes in the dephosphorylation of phosphotyrosine, requires expensive equipment, laborious preparation of samples, and cannot be used to monitor the activity of an enzyme on a continuous basis.
In spite of the various approaches described above, to date, there is no method for assaying PTP activity that is highly sensitive, fast, continuous, and relatively easy to perform. Thus, the development of a tool that can meet these requirements is highly desirable.